Estrogen receptor-alpha (ERalpha) is a ligand dependent transcription factor that is a therapeutic target for inhibition in breast cancer therapy. The transcriptional activity of ERalpha, and hence the ability of activated ERa to promote breast cancer proliferation is dependent upon interactions with transcriptional modulatory proteins termed coregulator proteins. Elevated expression of the growth factor membrane receptor HER2/neu is associated with breast cancer progression. HER2/neu activation is also linked to hyperactivation and hyper-phosphorylation of ERalpha in breast cancer leading to resistance to the antiestrogen tamoxifen. However the precise phosphorylation sites and mechanisms that link ERalpha and coregulators with HER2/neu signaling have not been defined. This proposal hypothesizes that elevated HER2/neu signaling in breast cancer leads to specific changes in ERalpha phosphorylation that alter the gene-specific recruitment of coregulators to ERalpha regulated genes. These HER2/neu induced changes in ERalpha phosphorylation are proposed to, in turn, promote breast cancer proliferation. Specific Aim 1 will identify the specific coregulators that are recruited to different classes of ERalpha responsive promoters, determine the effects of HER2/neu signaling on ERalpha and coregulator recruitment to these promoters, and determine whether the specific coregulators that were recruited to each gene promoter are required for expression of each gene. Specific Aim 2 will determine how HER2/neu induced changes in ERalpha phosphorylation regulate ERalpha interaction with coregulators and promoter recruitment, and determine the effect of these phosphorylation changes on ER- regulated gene expression and breast cancer cell proliferation. Understanding how phosphorylation of specific sites in ERalpha contributes to gene transcription through coregulator recruitment will provide mechanistic framework for understanding how current and future ERalpha-directed therapies function at the level of gene transcription. With reference to public health, this study will identify how two important proteins that cause breast cancer growth (estrogen receptor alpha (ERalpha) and HER2/neu) are linked to one another. This information will be important when designing breast cancer therapies that target both these proteins with the hope of improving therapies for breast cancer. [unreadable] [unreadable] [unreadable]